间尼索地平在细胞色素P450酶中的代谢及其代谢产物研究

发布时间：2018-06-23 08:18

本文选题：药物代谢 + 间尼索地平　；参考：《河北医科大学》2014年硕士论文

【摘要】：间尼索地平（m-nisoldipine）作为新型二氢吡啶类钙离子通道拮抗剂，主要用于高血压和心脏病的防治。以往研究表明其多以代谢物形式排出体外。肝脏作为药物代谢的重要器官，是多数Ⅰ相和Ⅱ相反应的发生场所，细胞色素P450酶是参与药物代谢和生物活化的主要酶类，了解药物与CYP450酶的作用关系能够为药物作用机制的研究及联合用药提供理论依据。本试验利用液质联用（HPLC-MS/MS）技术，采用人肝微粒体温孵、CYP450重组酶温孵及化学抑制剂试验，对参与间尼索地平生物转化的CYP450酶亚型进行鉴别。对灌胃给药大鼠组织中的间尼索地平及其代谢产物进行分析，为进一步理解其作用机制提供依据和技术支持。采用大鼠肝微粒体体外温孵及大鼠体内代谢的方法，初步探究间尼索地平Ⅱ相代谢中的葡萄糖醛酸结合反应。对间尼索地平对映体在人肝微粒体中的酶促反应动力学进行研究，优化反应条件，并比较两者的酶促反应动力学参数。第一部分间尼索地平在人源CYP450酶各亚型中的代谢研究目的：通过人肝微粒体温孵、CYP450重组酶温孵和化学抑制剂试验，对参与间尼索地平生物转化的CYP450酶亚型进行鉴别。方法：将温孵样品分别在EMS-IDA-EPI、 MRM-IDA-EPI和PREC-IDA-EPI模式下进行检测，分析反应生成的间尼索地平代谢产物。人肝微粒体温孵试验，将间尼索地平与人肝微粒体进行体外温孵，，应用HPLC-MS/MS技术检测代谢产物，获得二级质谱碎片信息，推测代谢产物结构。化学抑制剂试验，CYP450特异性抑制剂与人肝微粒体温孵30min后加入间尼索地平，一定时间后测定体系中代谢产物生成量，并与未加入抑制剂的阴性对照溶液比较。 CYP450重组酶温孵试验，将间尼索地平与CYP450重组酶进行体外温孵，寻找代谢产物并比较各亚型酶中间尼索地平的代谢情况。结果：在人肝微粒体温孵体系中共发现8种代谢产物，其中间尼索地平氧化脱氢、3位侧链羟基化及两者结合的3种代谢产物生成量远高于其他5种代谢产物。化学抑制剂试验中发现，盐酸噻氯匹定（CYP2C19抑制剂）能够明显抑制3位侧链羟基化产物生成，而酮康唑（CYP3A4抑制剂）对氧化脱氢产物的抑制作用明显。 CYP450重组酶试验中发现，CYP2C19和CYP3A4对间尼索地平的催化活性明显高于其他4种亚型，CYP2C19主要催化生成羟基化产物，CYP3A4对氧化反应的催化能力强。结论：化学抑制剂试验及CYP450重组酶试验结果一致表明，CYP2C19和CYP3A4是人肝微粒体中参与间尼索地平生物转化的主要酶亚型。第二部分HPLC-MS/MS法研究大鼠组织中间尼索地平及其代谢产物目的：通过对大鼠6种组织样品中间尼索地平代谢产物的分析，总结药物代谢规律，为进一步阐明其作用机制提供依据。方法：雄性SD大鼠灌胃给予间尼索地平1h后，取心、肝、脾、肺、肾和脑组织，加入生理盐水制成匀浆，经乙醚萃取后应用HPLC-MS/MS对样品进行分析。结合本实验室前期研究中已经进行结构确证的代谢产物，通过比较保留时间和二级质谱碎片信息推测所测得代谢产物的结构。结果：在6种组织样品中均检测到间尼索地平的代谢产物，其中在肝组织中发现9种代谢产物，且含量较高；在脾和肾组织中发现5种代谢产物；在心和肺组织中发现4种代谢产物；在脑组织中仅检测到2种代谢产物且含量很低。结论：间尼索地平在生物体内极易发生生物转化而生成代谢产物，肝脏是其代谢的主要场所，代谢途径主要为吡啶环氧化脱氢、吡啶环3位侧链羟基化及酯键的水解。第三部分间尼索地平Ⅱ相代谢葡萄糖醛酸化反应的初步探究目的：通过对大鼠肝微粒体体外温孵样品和胆汁、尿液的分析，初步探究间尼索地平Ⅱ相代谢的葡萄糖醛酸化反应。方法：向体系中加入UDPGA、丙甲菌素、MgCl2等辅酶，将间尼索地平与大鼠肝微粒体进行体外温孵，大鼠灌胃给予间尼索地平后收集36h内胆汁和尿液。应用HPLC-MS/MS技术对生物样品进行分析，在EMS-IDA-EPI、MRM-IDA-EPI和PREC-IDA-EPI模式下检测间尼索地平的葡萄糖醛酸代谢产物。结果：通过与空白样品比较，上述样品中未能检测到葡萄糖醛酸代谢产物，胆汁和尿液中只检测到Ⅰ相代谢物。结论：间尼索地平仅二氢吡啶环上仲胺含活泼H可与葡萄糖醛酸结合，但吡啶环极易氧化失去活泼H，同时试验中的反应条件、液相色谱-质谱条件也可能影响试验结果。因此，在今后的试验中，应尽可能优化试验条件，以进一步探明间尼索地平的葡萄糖醛酸结合反应。第四部分间尼索地平对映体在人肝微粒体中的酶促反应动力学研究目的：研究间尼索地平对映体在人肝微粒体中的酶促反应动力学，计算其酶促反应动力学参数，并对R型和S型间尼索地平的酶促反应动力学过程进行比较。方法：以尼莫地平为内标，Sapphire C18色谱柱（4.6mm×150mm，5μm），乙腈-0.1%甲酸水（90:10），等度洗脱，电喷雾离子源（ESI源），负离子模式下MRM监测，分别测定人肝微粒体体外温孵后R型、S型间尼索地平含量。采用Linewrave-Burk作图法处理数据，计算酶促动力学参数Km和Vmax。结果：间尼索地平对映体在人肝微粒体中最佳温孵时间为30min，蛋白浓度为1mg·mL-1，底物浓度为25μmol·L-1。R-间尼索地平Km=79.20μmol·L-1，Vmax=2.727μmol·(min·mg protein)-1；S-间尼索地平Km=83.8μmol·L-1，Vmax=2.892μmol·(min·mg protein)-1。结论：该方法简单、可靠，适用于间尼索地平的体外代谢研究，R型和S型间尼索地平的酶促反应动力学过程无明显差异。
[Abstract]:M-nisoldipine is a new two hydropyridine calcium channel antagonist, which is mainly used in the prevention and treatment of hypertension and heart disease. Previous studies showed that it was expelled in the form of metabolites. The liver, as an important organ for drug metabolism, is the place where the majority of phase I and II are opposite. Cytochrome P450 enzyme is involved in the drug. Understanding the relationship between drugs and CYP450 enzymes can provide a theoretical basis for the research of drug action mechanism and the combination of drugs.
In this experiment, we used liquid mass spectrometry (HPLC-MS/MS) technique to identify the CYP450 enzyme subtypes of the biotransformation of nisodipine by using the temperature hatching of human liver particles, incubation of CYP450 recombinant enzyme and chemical inhibitor test, and the analysis of the nisodipine and its metabolites in the rat tissues of gavage to further understand the mechanism of its action. To provide basis and technical support. The methods of incubating rat liver microsomes in vitro and metabolism in rats were used to explore the glucuronic acid binding reaction in the phase II metabolism of nisoxdipine. The enzyme reaction kinetics of the enantiomer in human liver microsomes was studied, the reaction conditions were optimized, and the enzymatic reaction of the two was compared. Dynamic parameters.
Part one metabolism of nisodipine in human CYP450 subtypes
Objective: to identify CYP450 enzyme subtypes involved in the bioconversion of nicardipine through incubation of human liver microparticles, incubation of CYP450 recombinase and chemical inhibitor test.
Methods: the samples were incubated in EMS-IDA-EPI, MRM-IDA-EPI and PREC-IDA-EPI mode respectively, and the metabolites of the reaction were analyzed.
The temperature incubation experiment of human liver microparticles was incubated with the human liver microsomes in vitro, and the metabolites were detected by HPLC-MS/MS technique, and the two level mass spectra of mass spectra were obtained, and the structure of the metabolites was speculated.
The chemical inhibitor test, the CYP450 specific inhibitor and the body temperature of the human liver were incubated with the temperature of the human liver microparticle for 30min. After a certain time, the amount of metabolites in the system was measured and compared with that of the negative control solution, which was not added to the inhibitor.
CYP450 recombinant enzyme incubated in vitro and incubated between nisodipine and CYP450 recombinant enzyme in vitro to find metabolites and compare the metabolism of nisopodipine in the middle of each subtype.
Results: 8 kinds of metabolites were found in the body temperature Incubating System of human liver particles, including the oxidative dehydrogenation of nippine, the 3 side chain hydroxylation and the combination of the 3 kinds of metabolites were much higher than the other 5 kinds of metabolites.
In the chemical inhibitor test, it was found that tilopidine hydrochloride (CYP2C19 inhibitor) could obviously inhibit the formation of 3 side chain hydroxylation products, while ketoconazole (CYP3A4 inhibitor) inhibited the oxidative dehydrogenation products.
In the CYP450 recombinant enzyme test, it was found that the catalytic activity of CYP2C19 and CYP3A4 on the inter nisodipine was significantly higher than that of the other 4 subtypes. CYP2C19 mainly catalyzed the formation of hydroxylation products, and CYP3A4 had a strong catalytic ability to the oxidation reaction.
Conclusion: the results of chemical inhibitor test and CYP450 recombinant enzyme test show that CYP2C19 and CYP3A4 are the main subtypes of biotransformation of nisisodipine in human liver microsomes.
The second part is to study the relationship between nndipine and its metabolites in rat tissues by HPLC-MS/MS.
Objective: through the analysis of the metabolites of the 6 tissue samples in the middle of the rat, the regulation of drug metabolism was summarized and the basis for further clarifying the mechanism of its action was provided.
Methods: after the male SD rats were fed with neldipine 1H, the heart, liver, spleen, lung, kidney and brain tissue were taken into the homogenate, and the homogenate was made by adding physiological saline. After the ether extraction, the samples were analyzed with HPLC-MS/MS. The metabolites confirmed by the structure in the previous study in this laboratory were compared, and the retention time and the two grade mass spectrometry fragment letter were compared. The structure of the metabolites was measured by the interest speculates.
Results: the metabolites of interdipine were detected in 6 tissue samples, of which 9 metabolites were found in the liver tissue with high content; 5 metabolites were found in the spleen and kidney tissue; 4 metabolites were found in the heart and lung tissues; only 2 metabolites were detected in the brain tissue and the content was very low.
Conclusion: biotransformation of nxdipine in organism is very easy to produce biotransformation and produce metabolites. The liver is the main place for its metabolism. The main metabolic pathways are oxidative dehydrogenation of pyridine ring, 3 side chain hydroxylation of pyridine ring and hydrolysis of ester bond.
A preliminary study of third phase naloxidipine II metabolism glucuronization reaction
Objective: To explore the glucuronization reaction of phase II metabolism of nicoslodipine by analyzing the samples and bile from rat liver microsomes incubated in vitro.
Methods: UDPGA, methyloxin, MgCl2 and other coenzyme were added into the system to incubate the inter nosdipine with rat liver microsomes in vitro. The rats were given the bile and urine in 36h after gavage, and the biological samples were analyzed by HPLC-MS/MS, and the nnso was detected in the EMS-IDA-EPI, MRM-IDA-EPI and PREC-IDA-EPI modes. A glucuronic acid metabolite of the horizon.
Results: glucuronide metabolites were not detected in the above samples compared with blank samples. Only phase I metabolites were detected in bile and urine.
Conclusion: the active H in the two hydropyridine ring is only active with glucuronic acid, but the pyridine ring is very easy to oxidize and lose active H. At the same time, the conditions of the reaction in the test and the liquid chromatography-mass spectrum condition may also affect the test results. Therefore, in the future experiment, we should optimize the test conditions as far as possible to further explore the intern Nile. The glucuronic acid binding reaction of the horizon.
Kinetics of enantiomeric reaction of fourth nisodipine enantiomers in human liver microsomes
Objective: To study the enzyme catalyzed kinetics of nisedipine enantiomers in human liver microsomes, to calculate the kinetic parameters of the enzyme reaction, and to compare the kinetics of the enzymatic reaction between R and S type nisodipine.
Methods: with nimodipine as the internal standard, Sapphire C18 column (4.6mm x 150mm, 5 mu m), acetonitrile -0.1% formate water (90:10), ISO elution, electrospray ion source (ESI source) and MRM monitoring under negative ion mode, the content of R and S type nxodipine was measured in vitro after incubating in human liver microsomes, and the data were processed by Linewrave-Burk mapping method and enzymatic method was used to calculate enzyme promotion. Dynamic parameters Km and Vmax.
Results: the best incubation time of the enantiomers in human liver microsomes was 30min, the concentration of the protein was 1mg mL-1, the concentration of the substrate was 25 mu mol / L-1.R-, Km=79.20 Mu mol / L-1, and Vmax=2.727 micron mol.
Conclusion: the method is simple, reliable and suitable for the in vitro metabolism study of nicardipine. There is no significant difference in the enzymatic reaction kinetics between nimodipine and type R S.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965;O657.63

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